Apparatuses designed for cultivation of microbial organisms or eukaryotic cells, known as bioreactors or fermentors, have been used for production of various biological or chemical products in the pharmaceutical, biotechnological and beverage industry. A typical bioreactor includes a vessel containing culture medium in a sterile environment that provides the various nutrients required to support growth and production of the homogeneous biological agents of interest. Effective cell culture process requires appropriate supplies of nutrient substances, such as glutamine, glucose, and other medium components; and gas, such as oxygen and carbon dioxide, for growing cells in a bioreactor. In addition, timely control of physiological conditions, such as appropriate pH, temperature, and osmolality is required for mass production of microbial and cell cultures. To conduct all of these control schemes the analysis of these bioprocessing parameters is required and performed by in-situ sensor or on-line/off-line sampling and analysis. Imperative utilization of biosensors has acquired paramount importance in the field of not only bioprocessing but also in food safety standards, defense, security, and environmental monitoring. This has led to an exponential growth of related R&D efforts around the world in recent years. However, on-line/off-line analysis remains the most popular and practical approach for bioprocessing because of the wide availability of off-line analytical instruments. During a microbial or cell culture process, therefore, aseptic withdrawal of a culture broth sample that is representative of the overall microbial or cell culture condition is critical for monitoring the performance of the cell culture or fermentation process and for controlling the process or troubleshooting of any process problems. Conventional sample withdrawal from a bioreactor, fermentor, or medium holding vessel, however, is typically performed by a series of manual operations, including purging of the sampling line, connecting a sample device aseptically to the line, removing the sample from the bioreactor, and closing the line. The purge step is usually required at the beginning of each sampling step to push the residual sample in the sampling line from the previous sampling into a waste reservoir. The conventional sample withdrawal procedure results in the waste of the sample held in the main sampling line, and requires an additional step to switch the sampling line between the waste reservoir and the actual sample container. Most patents including U.S. Pat. Nos. 8,281,672, 7,848,848, 7,213,474, 4,942,770, 5,525,301, etc. taught the use of various automated aseptic sampling devices for bioreactors. All of them have the sampling uptake point inside the vessel in a stationary position. Therefore, it would always have the sample remain in the entire or partial portion of the line depending on the configuration of devices and sampling procedures after a given amount of sample has been collected. The remaining sample requires to be flushed or purged to the waste for the next segment of fresh sampling. This innovation develops a sampling device not only to collect the sample aseptically in any quantity of interest, but to also not require flushing the residual sample to the waste.
As mentioned, glucose is the most important carbon source for a cell or microbial culture. It is therefore the key component of the culture medium most commonly used as an indicator for controlling the culture. Glucose concentration of microbial or cell culture is most commonly measured off-line by an enzymatic glucose analyzer or a glucose analyzer of which the measurement includes glucose and many other biochemicals in the culture medium such as pH, Lac, Gln, MH4, PO2, PCO2, Na+, K+, PO4, Gly, Ca++. Most of these analyzers and consumables are expensive, particularly, when it is configured for automatic measurement. Lately, the use of a manual inexpensive blood glucose meter for glucose measurement in microbial and cell cultures has gained popularity because of its low cost, small sample volume required and fast response time. However, it is manually operated and requires to be calibrated because its accuracy is medium-dependent on each medium used in the cultures.
This disclosure provides a risk-free and accurate sampling method to automatically collect the fluid sample from a bioreactor in an exact amount as low as <1 ml, no waste of any fluid, and no requirement of purging or flushing. It is also coupled with a low cost automatic glucose monitoring system to measure the glucose concentration of the collected sample automatically, accurately and economically.